Fatty acids increase glucose uptake and metabolism in C2C12 myoblasts stably transfected with human lipoprotein lipase

Abstract

Cellular effects of FFA might differ from those of lipoprotein triglyceride (TG)-derived fatty acids (TGFA). The aim of the current study was to examine the relationship between lipoprotein lipase (LPL) expression, TGFA, or FFA availability and glucose metabolism in the absence of insulin in C2C12 myoblasts. Control myoblasts or myoblasts stably transfected with human lipoprotein lipase (C2/LPL; 15-fold greater LPL activity) were incubated for 12 h in fetal bovine serum-free medium in the absence or presence of Intralipid-20. Intracellular retention of labeled medium glucose was assessed in a subset of experiments. In the presence of Intralipid, medium glucose disappearance was increased in C2/LPL cells but not in control cells. In both cell types, glucose label retention in cellular TG was increased in the presence of Intralipid; incubation with albumin-bound oleate produced similar results. In the presence of Intralipid, the LPL hydrolytic inhibitor tetrahydrolipstatin blocked excess glucose retention in cellular TG but did not significantly decrease glucose disappearance in C2/LPL cells. Changes in glucose transport or hexokinase II did not explain the altered glucose disappearance in C2/LPL cells. Our results suggest that LPL overexpression in these cells leads to chronic metabolic adaptations that alter glucose uptake and retention.

Fig. 1.

A: heparin-releasable lipoprotein lipase (LPL) activity in control and C₂/LPL myoblasts in the absence and presence of tetrahydrolipstatin (THL). The minimum concentration of THL found to completely inhibit LPL in both cell types (50 μM) was used in subsequent THL experiments; n = 3 for each concentration of THL. B: changes in medium free fatty acid (FFA) concentration when control and C₂/LPL cells were provided with TG+ or THL medium. Net appearance of medium LPL-generated FFA under TG+ conditions, reflected by the difference between TG+ and THL curves, was evident for both control (P = 0.05) and C₂/LPL (P < 0.001) cells. Values are expressed as means ± SE; n = 5.

Fig. 2.

A: densitometry of Oil red O-stained micrographs of control and C₂/LPL cultures after incubation with TG−, TG+, and FFA media for 12 h. Values are expressed as means ± SE; n = 3. ANOVA; *P = 0.008 vs. TG−; ♦P < 0.001 vs. TG−. B: medium glucose disappearance over the 12-h incubation in control and C₂/LPL cultures under TG−, TG+, and FFA conditions. Values are expressed as means ± SE. ANOVA; *P < 0.001 vs. TG−; §P = 0.002 vs. TG−.

Fig. 3.

Retention of medium glucose label in cell lipid subfractions in control (A) and C₂/LPL cells (B) under TG−, TG+, and FFA conditions. Values are expressed as means ± SE. For all conditions, n = 4. Multivariate ANOVA; *P = 0.03 vs. TG−; ♦P < 0.001 vs. TG− and TG+; §P = 0.003 vs. TG−. TG, triglyceride; DAG, diacylglycerol; MAG, monoacylglycerol; PL, phospholipid; CE, cholesterol ester; Chol, cholesterol.

Fig. 4.

Effects of incubation with TG+ medium or THL medium containing the LPL catalytic site inhibitor THL on glucose disappearance (A), glucose label retention in cell TG (n = 4; B), and neutral lipid accumulation by Oil red O staining (n = 3; C). Values are expressed as means ± SE. *P = 0.004 vs. TG+.

Fig. 5.

Representative Western blots for phospho (p)-Akt and total (tot) Akt (n = 5), phospho-AMP-activated protein kinase (AMPK) and total AMPK (n = 3), GLUT1 (n = 3), GLUT4 (n = 6), and hexokinase II (HKII; n = 4) in control and C₂/LPL cells following lipid treatments. A positive control coincided with the upper band of the observed GLUT1 doublet. For GLUT1 and GLUT4, membranes were stripped and reprobed for β-actin as a loading control. Densitometry of Western blots is shown at right. Akt phosphorylation (P = 0.01) and HKII expression (P = 0.03) were significantly lower in C₂/LPL cells than in controls.